Hemophilia B (HB) is an inherited bleed diathesis due to absence of functional coagulation F. IX. Gene transfer is an appealing approach to HB since small increases in plasma F.IX. Gene transfer is an appealing approach to HB since small increases in plasma F.IX improve the clinical course and tight control of transgene expression is unnecessary for clinical benefit. Efficacy and dose-finding can be extrapolated from experiments in animal models. A human trial assessing the safety of intramuscular (IM) administration of adeno-associated virus (AAV) vector with a CMV promoter encoding F.IX was developed 2-3 years ago, but new tools are now available that will likely improve the current vector. We propose three strategies for enhanced transgene expression and improved patient safety in the clinical trial. First, we will make a vector with a muscle-specific promoter to reduce the risk of immune response against the monocyte derived F.IX. Second, we will assess if vector injection into muscles rich in slow fibers ( soleus of the leg) results in enhanced murine transgene expression Third, since AAV-1 capsid provides superior gene transfer compared to the AAV-2 capsid in the current vector, we will test whether an AAV-1 vector produces better gene transfer than the AAV-2 in RAG-1 mice. Those modifications that result in enhanced transgene expression will be used to create a novel vector for a new trial of AAV-F.IX for IM administration. We will enroll 9 adult males with severe HB in a dose escalation safety trial. Three subjects will be enrolled in each of 3 dose cohorts. We will monitor subjects for local and systemic toxicity, antibody formation against the viral capsid Ag and F.IX and body fluids for the presence of vector sequences. Clinical endpoints will include changes in aPTTs, F.IX levels and patterns of factor concentrate use. After safety is demonstrated, we will establish if re-treatment with an AAV vector is useful, by injecting the new vector into subjects previously treated with AAV-CMV-hF.IX who have demonstrated low transgene expression.